As infection with Pasteurella multocida is common in cattle and buffalo, a monoclonal antibody based sandwich ELISA kit was developed for its rapid and easy detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of hemorrhagic septicemia diagnosis recommended by the WHO expert committee. HS-1, a Pasteurella multocida type B specific monoclonal antibody developed in mice was used as tracing antibody to capture P. multocida serotype B:2 in a sandwich ELISA. The test was standardized for whole killed bacterial cell, sonicated and the LPS antigens of P. multocida type B:2. An anti-pasteurella hyper immune serum raised in rabbit acted as the coating antibody was selected since it was previously shown to be a major immunogen during P. multocida infection in rabbits and contain antibodies against several conserved epitopes. The sensitivity of the sandwich ELISA determined with ELISA well module (8x2) for whole killed bacterial cells, and with two fold serial dilutions of an antigen (12 steps and in triplicate) for sonicated and LPS antigen, were 1.6X1011 cfu/ml, 385 ng/ml and 17.4 mg/ml respectively. Specificity, evaluated against the cultured P. multocida type A antigen of bovine strain, was 100%. The coefficient of variation for sonicated and the LPS antigens calculated from intra and inter plate for same-day and inter-day tests were found within 20% indicating good reproducibility with few exceptions when CV varied more than 20%.
Trang 1Original Research Article https://doi.org/10.20546/ijcmas.2019.805.128
Development of a Specific Monoclonal Antibody based Sandwich ELISA for
Rapid Detection of Haemorrhagic Septicemia in Bovine Blood
Ragini Hazari 1* , Arvind Kumar 1 and Sonu Sharma 2
1
Department of Veterinary Microbiology, 2 Department of Veterinary Pathology, Lala Lajpat
Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India
*Corresponding author
A B S T R A C T
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 8 Number 05 (2019)
Journal homepage: http://www.ijcmas.com
As infection with Pasteurella multocida is common in cattle and buffalo, a monoclonal
antibody based sandwich ELISA kit was developed for its rapid and easy detection The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of hemorrhagic septicemia diagnosis recommended by the
WHO expert committee HS-1, a Pasteurella multocida type B specific monoclonal antibody developed in mice was used as tracing antibody to capture P multocida serotype
B:2 in a sandwich ELISA The test was standardized for whole killed bacterial cell,
sonicated and the LPS antigens of P multocida type B:2 An anti-pasteurella hyper
immune serum raised in rabbit acted as the coating antibody was selected since it was
previously shown to be a major immunogen during P multocida infection in rabbits and
contain antibodies against several conserved epitopes The sensitivity of the sandwich ELISA determined with ELISA well module (8x2) for whole killed bacterial cells, and with two fold serial dilutions of an antigen (12 steps and in triplicate) for sonicated and LPS antigen, were 1.6X1011 cfu/ml, 385 ng/ml and 17.4 mg/ml respectively Specificity,
evaluated against the cultured P multocida type A antigen of bovine strain, was 100%
The coefficient of variation for sonicated and the LPS antigens calculated from intra and inter plate for same-day and inter-day tests were found within 20% indicating good reproducibility with few exceptions when CV varied more than 20% In some instances,
CV values as high as 23% and 25% were recorded for whole killed bacterial cell CV values up to 25% indicated repeatability of the ELISA and higher OD values for whole killed bacterial cells did not record high standard deviations (SD) Therefore, the ELISA showed repeatability of the test for all three types of antigens Furthermore, more
expression of the P multocida type B:2 specific PCR in comparison to P multocida type
B and P multocida PCR in early phase of pathogenesis of the disease was detected and showed the greater analytical sensitivity and specificity to identify bovines infected with P multocida The results showed that this sandwich ELISA, with good specificity, sensitivity
and simplicity, would be a useful assay for an early clinical diagnosis of HS The ELISA can be performed directly on infected blood in modestly equipped laboratory, manned by semi skilled personnel
Trang 2Introduction
Hemorrhagic Septicemia (HS) is a major
disease of cattle and buffaloes occurring as
epizootics in many Asian and African
countries It is caused by specific serotypes
within the bacterial species of Gram negative
coccobacillary pathogen Pasteurella multocida
viz B:2 and B:5 in Asian counties and type
E:2 in African countries (Carter and De Alwis,
1989) On the basis of capsular antigens, P
multocida has been classified into five
serotypes, namely A, B, D, E and F (Carter,
1955), whereas on the basis of somatic
antigens, it has been classified into sixteen
serotypes (Heddleston et al., 1972) According
to current classification, the family
Pasteurellaceae includes a large group of
Gram negative bacteria that are
chemo-organotrophic, facultatively anaerobic and
fermentative in nature (Mutters et al., 1989) It
is a disease of utmost economic importance
particularly in Asia due to large population of
buffaloes The case fatality rate and
susceptibility to HS are higher in buffaloes
than in cattle (Benkirane and De Alwis, 2002)
HS has been estimated to cause huge economic
losses in India, to the tune of Rs 225 millions
(Singh et al., 2008) However, the losses are
expected to be much greater than that have
been reported because of poor reporting and
surveillance systems
Once clinical signs appear, case fatality is
nearly 100% Variable number of immune
carriers is present in animal populations,
particularly in endemic areas They may be
latent carriers, where the organisms are
lodged in the tonsils, or active carriers, where
organisms are detectable in the naso-pharynx
The long lasting carrier status may escape the
notice of the animal health authorities but
may be of considerable economic significance
(De Alwis, 1981) HS is, typically a
septicemic disease that develops following
release of endotoxin from dead bacterial cells
It has an affinity for respiratory tract mucus
membrane (Lettellier et al., 1991) and a better
affinity for non-ciliated respiratory epithelial cells (Pijoan and Trigo, 1990)
The pathogenic components of P multocida
include capsule, endotoxin and outer membrane proteins which have been reported
to be the virulence factors responsible for immuno-pathological changes (Boyce and
Adler, 2006; Singh et al., 2011)
Regular vaccination at six months interval, before onset of rainy season and beginning of winter, is a major control measure for prevention of HS Various killed vaccines, for example, broth bacterin, aluminum hydroxide precipitated vaccine and the oil adjuvant vaccine, are commonly used for
immunization against HS (Tasneem et al.,
2009) Short duration of immunity of only 4-6 months is a major limitation of the conventional killed bacterial vaccines Oil adjuvanted vaccine induces immunity of little longer duration of about 9 months, yet far lower than that needed (De Alwis, 1992a; OIE, 2012) For improving the duration of immunity, live vaccine with aroA mutant has been experimentally developed and a live
vaccine using deer strain (P multocida B:3,4)
has also been reported (Verma and Jaiswal, 1998) The live vaccine of deer strain is used
only in Myanmar (De Alwis, 1999)
It is a common practice that antibiotics are used to treat the diseased animals HS is a septicemic disease and the animal dies of
endotoxic shock (Horadagoda et al., 2001)
The bacteria multiply fast in the blood of diseased animals and die Release of LPS (endotoxin) from dead bacterial cell causes endotoxic shock and death of the animal Treatment with antibiotics after appearance of clinical signs (fever and recumbency), may hasten the rate of death of bacterial cells in the blood This may further exacerbate the
Trang 3condition and hasten the death of animal (De
Alwis, 1999) Inadequate vaccination based
control programme and difficulties of
antibiotic treatment at later stage of the
disease, put emphasis on development of not
only a specific and a sensitive diagnostic test
but use of the test should also reduce time
taken for diagnosis (time spent on collection
and reaching of samples to the lab as well as
time needed to complete the test procedure)
The test of high sensitivity would detect
smaller amount of antigen in clinical samples
and therefore may have capacity for diagnosis
at an early stage, before the onset of clinical
symptoms Point-of-care diagnostics would
reduce the time taken for diagnosis
Therefore, a highly sensitive point-of-care
diagnostics is an ideal test
The polymerase chain reaction (PCR) assay
has been reported for amplification of P
multocida gene, P multocida type B and P
multocida B:2 (Brickell et al., 1998 and
Townsend et al., 1998) PCR is a rapid,
specific and highly sensitive test for
confirmation of HS but it requires
sophisticated laboratory and highly skilled
manpower Therefore, PCR could not be
developed as point-of-care diagnostic test
The PCR can detect even very little amount of
target DNA in the infected blood Despite its
very high sensitivity, time taken for
collection, dispatch and receipt of samples in
laboratory and time taken for completion of
procedure of PCR defeats its use for
diagnosing the disease at an early stage
Attempts have been made to develop simple
agglutination test as point-of-care diagnostic
test Use of agglutinating monoclonal
antibody coated coloured latex beads based
agglutination has given desired sensitivity and
specificity for clinical diagnosis of the disease
(Pankaj Kumar and Arvind Kumar, Indian
Patent Application No 767/DEL/2015 filed
test is not an objective test and therefore, an inexperienced operator may cause error of judgment
It is, therefore necessary to develop simple, accurate and rapid diagnostic test that can be carried out by semi-skilled laboratory personnel even in the modestly equipped laboratories at block or district level ELISA has been successfully used for diagnosis of various bacterial and viral diseases (OIE, 2012) The test can also be converted as a kit for use as point-of-care diagnostics (Anon,
2004, Lister et al., 2012) Dawkins et al.,
(1990) developed an ELISA for detection of
P multocida B:2 However, the test has been
described as a useful tool for screening of bacterial isolates and is based on polyclonal serum Use of polyclonal serum raises a doubt
on specificity of the test Perusal of scientific literature did not show the use of test as field diagnostic test for HS
Monoclonal antibodies have been described
as bio-reagent of unmatched specificity that can distinguish even very closely related micro-organisms and of very high sensitivity because of no undesired cross reactive back ground reactions in ELISA Polyclonal serum may contain antibodies against several conserved epitopes and therefore, polyclonal serum shows cross reactivity in ELISA The conserved epitopes are generally immunodominant epitopes and the epitopes discriminating closely related micro-organisms are immunosubdominant epitopes Low amount of antibody against such immunosubdominant epitopes(s) would lower sensitivity of the test The specificity and sensitivity of the ELISA test are expected to
increase many folds with P multocida B
specific monoclonal antibody and is therefore suitable for early diagnosis of the disease The test could also be converted in to point-of-
care diagnostic test A P multocida B specific
monoclonal antibody has been developed in
Trang 4our laboratory (Pankaj Kumar, 2014)
Considering the fact that the matrix of clinical
sample would be the infected blood, a
sandwich ELISA would be more suited for
the purpose The ELISA plate wells coated
with anti-pasteurella multocida polyclonal
serum would selectively capture the P
multocida antigen present in the sample and
then P multocida B specific monoclonal
antibody would bind only to captured P
multocida type B bacteria/antigens This
study is therefore targeted to Development
and optimization of monoclonal antibody
sandwich ELISA for diagnosis of
Hemorrhagic Septicemia of bovines and
evaluation of the test for clinical diagnosis of
Hemorrhagic Septicemia
Materials and Methods
The bacteria
Buffalo calf blood experimentally infected
with P multocida B:2 P52 (Vaccine strain)
was obtained from Haryana Veterinary
Vaccine Institute (HVVI), Hisar The
bacterial stock was prepared by streaking the
infected blood on 5% sheep blood agar and
incubated overnight at 35-36ºC The bacterial
growth on the Petri plate was agar washed
and aliquoted in 50% brain heart infusion
broth (BHI broth) and glycerol The aliquots
were stored at -20ºC The bacteria were
revived as and when required, by streaking on
5% sheep blood agar and incubated overnight
at 35-36ºC For short duration storage of 4-5
days, the bacterial cultures were kept at +4ºC
Buffalo calf blood experimentally infected
with P multocida B:2 P52 (Vaccine strain)
was obtained from Haryana Veterinary
Vaccine Institute (HVVI), Hisar P multocida
type A (bovine strain) was obtained from Indian
Veterinary Research Institute, Izatnagar and
was maintained and cultured Anti-mouse
HRPO conjugate raised in rabbit was
purchased from Sigma Aldrich Co., (cat No.9044) and referred as “The Conjugate” Anti-pasteurella multocida monoclonal antibody referred as “HS-1 Mab” was produced in the department during the doctoral research of Pankaj Kumar (2014) by the hybridoma secreting the monoclonal
antibody and raised in vivo in ascites fluid of
mice, anti- pasteurella multocida whole bacterial cell hyper immune serum named as
“The coating antibody” and raised in rabbit
P multocida type B specific PCR primers
KTSP61 and KTT72 were commercially synthesized from Sigma-Aldrich Chemical Pvt Ltd., Bangalore, India
Experimental animals
Male Swiss albino mice aged 8-10 weeks were procured from Disease Free Small Animal House, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar The
mice were caged, fed ad libidum and used
within 2 days of procurement Approval of Institutional Animal Ethics Committee for animal experimentation was granted vide VPHE/IAEC/88-108 dated 19/04/2014
monoclonal antibody sandwich ELISA for diagnosis of hemorrhagic septicemia
A monoclonal antibody based sandwich ELISA was developed and optimized as per the guidelines of OIE (2012) for assay development The test was standardized for: determination of optimum dilutions of the bio-reagents, repeatability, estimation of cutoff, analytical sensitivity and specificity and normalization of data The test was developed
to the extent that it could become fit for clinical diagnosis of experimentally produced
HS in mice Stocks of P multocida B:2 and
P multocida type A were raised and their
Purity, identity was done by using various
biochemical tests viz Catalase test, Oxidase
Trang 5test, Indole test, and confirmation was done
by using PCR with KTSP61:5’-
ATC-CGC-TAA-CAC-ACT-CTC-3’ (Tm= 55.00C) (F)
and KTT72:
5’-AGG-CTC-GTT-TGG-ATT-ATG-AAG-3’ (Tm= 61.90
C) (R) primers
Preparation of bacterial antigens
Whole killed bacterial cells antigen, sonicated
antigen and LPS antigen were prepared The
formaldehyde treated A 37% formaldehyde
solution was added to achieve 0.5%
concentration of formaldehyde in The P
multocida B:2 BHI broth A small volume of
5 ml was saved for estimation of colony
forming units (cfu) It was stored at +4OC
overnight and used next day for estimation of
cfu
The whole killed bacterial cell antigen
Of the 195 ml The P multocida B:2 BHI
broth of inactivated bacteria, approximately
50 ml broth was used It was pelleted by
centrifugation at 3000 rpm (swing out rotor,
Remi centrifuge model no R-4CDx
Laboratory centrifuge) Supernatant was
discarded and bacterial pellet was suspended
in 20 ml phosphate buffer saline (PBS-T) It
was stored at +4ºC till use
Estimation of colony forming units
Limiting dilution method was used (Prescott
et al., 2002) The dilution that produced
about 20-30 bacterial colonies on the Petri
plate was considered for estimation of cfu in
The P multocida B:2 BHI broth
The sonicated bacterial antigen
Approximately 100 ml of The P multocida
B:2 BHI broth was centrifuged as described
above Supernatant was discarded and pellet
was suspended in about 30 ml PBS-T The bacterial cells were disrupted by ultra sonication (ultra sonication model ultrasonicator-Micronix S-4000 micronix) as per standard procedure and the supernatant was saved and stored at -20oC till use This way sonicated bacterial antigen was prepared
Estimation of protein concentration of sonicated antigen
Estimation of protein concentration of sonicated antigen was done as per the method described by Bradford (1976) by using Coomassie Blue G-250 The absorbance of the sample at 595 nm was taken and the protein concentration was determined from the standard curve
The LPS antigen
Bacterial LPS antigen was prepared as
described by Pandian et al., (1999) A total of
45 ml of The P multocida B:2 BHI broth was
used for preparation of LPS antigen
Estimation of carbohydrate concentration
of LPS antigen
The amount of carbohydrate was estimated
for determination of analytical sensitivity of The ELISA test of LPS antigen It was done
by sulphuric acid-UV method using the regression equation as described by
Albalasmeh et al., (2013)
Assay development and validation
Sandwich ELISA was developed and optimized The ELISA plate wells were coated with the coating antibody The coating antibody captured the antigen Then antigen was detected by the HS-1 Mab The binding
of the HS-1 Mab with the captured antigen was detected by the conjugate and finally the test was developed with 3,3,5,5, Tetra Methyl
Trang 6Benzidine (TMB) The optical density of the
colour (OD value) was recorded in an ELISA
reader at filter wave length 450 nm The test
was standardized and optimized for all three
types of antigens (whole killed bacterial cell
antigen, sonicated antigen and the LPS
antigen)
For the sandwich ELISA, the wells of a 96
well flat bottom ELISA plate (Maxisorp,
Nunc) were coated with 50µl/well of
appropriate dilution of the coating antibody
prepared in coating buffer The plate was
covered and kept at +40C overnight Next day,
the plate wells were washed with washing
buffer (PBS-TT) and then discarded by flick
of wrist This was done 5X
The wells of the plate were blotted dry This
way washing and drying of the ELISA plate
wells were done Antigen was diluted in
diluent and appropriately diluted antigen was
added as 50µl/ well The plate was covered
with plate cover and then incubated 35-360C
for 1h The wells of the plate were again
washed and dried as described above
Appropriately diluted HS-1 Mab, was added
as 50µl/ well and ELISA plate incubated as
described above The plate wells were washed
as done above and appropriately diluted
conjugate was added as 50µl/ well The plate
was again incubated, washed and dried as
described above
To develop the test, TMB solution was added
as 50µl/ well The colour was allowed to
develop for 7-8 min and then the reaction was
stopped by acidification The stopping solution
was added 50µl/ well The OD values were
read at 450 nm wave length This is described
as, “The ELISA test.”
In each setup of the ELISA, appropriate
control was kept The controls were set up as
blank, negative and positive controls as
required in an experiment
Standardization of the test
Chequer board titration was performed for
standardization of the test This was done for determination of optimum dilutions of: 1- The coating antibody, 2- HS-1 Mab and 3- The conjugate The optimum dilutions were determined for 1:5, 1:50 and 1:100 dilutions of antigens
A 96 well ELISA plate (Nunc) was divided in
to four parts The coating antibody was fold diluted column-wise (1:125 to 1:1000, 4 steps) and the conjugate diluted two fold row-wise (1:200 to 1:6400, six steps) A dilution
two-of HS-1 Mab was added to wells two-of one two-of four parts of the plate The dilutions of HS-1 Mab used were 1:500, 1:800, 1:1000 and 1:1500 Layout of ELISA plate is shown in Figure 11 Combination of the most diluted coating antibody, HS-1 Mab, The conjugate and the antigen in a well showing appreciable colour development (at least, OD 0.2) taken
as optimum dilution of The coating antibody, HS-1 Mab and The conjugate
The repeatability studies
For testing the robustness of the test, repeatability study was performed The experiment was set up as described in OIE (2012) However, 24 replicates of each three
dilutions of an antigen (1:5, 1:10, 1:50) were
kept in the ELISA plate These way three plates were set up and the experiment was
repeated in similar manner on three different
days
Statistical analysis
Mean and standard deviations (SD) of OD values of the wells: intra-plate, inter-plate and inter-days were calculated in MS Excel 2007 From the values of mean and SD, coefficient
of variation (CV) was calculated by dividing
SD over mean (CV= SD/mean) The CV(s)
Trang 7were calculated for all the three dilutions of
all the three antigens
Estimation of cut off value
Cut off value is determined to establish
criterion for declaring a test positive or
negative In the study, mean + 3SD of OD
value negative control wells were taken as cut
off value The ELISA was set up with two
different types of negative control wells In
one type of negative control wells, only
diluent was added and no antigen was added
In the second type of negative control wells,
an antigen of P multocida B:2 was added but
an ascites fluid containing unrelated
monoclonal antibody was used The ELISA
test was performed with all three types of the
P multocida B:2 antigens The ELISA test
conditions were kept same as standardized for
a type of P multocida B:2 antigen For the
ascites of unrelated monoclonal antibody, the
dilution of the ascites fluid was kept similar to
the dilution of HS-1 Mab for that type of P
multocida B:2 antigen
Analytical sensitivity
Analytical sensitivity is the lowest amount of
analyte that can be detected by the test For
estimation of analytical sensitivity of The
ELISA standardized for all three types of
antigens The ELISA test was performed with
two fold serial dilutions of an antigen (12
steps and in triplicate) but for whole killed
bacterial cell antigen it was performed in the
ELISA well module (8X2) The highest
dilution of antigen recorded OD value equal
to the cut off value was taken as the ELISA
titre The dilution of the ELISA titre was used
to estimate the amount of an antigen in that
dilution The amount of antigen in stock of
whole killed bacterial cell, sonicated and the
LPS were estimated as described in sections
3.6.2.1a, 3.6.2.2a, 3.6.2.3a respectively
Amount of antigen in stock was divided by
the dilution of the ELISA titre to calculate amount of antigen in 50µl volume of the antigen dilution This way analytical sensitivity of the ELISA for all three types of antigen was established
Analytical specificity
Analytical specificity is the degree to which the assay does not cross react with other analyte Analytical specificity of The ELISA
test was established against P multocida type
A antigen of bovine strain and the ELISA test
was performed with a change that P
multocida type A antigen was used in place of
P multocida B:2 antigen The ELISA was
done with all three types of the antigens (whole bacterial cell killed, sonicated and the
LPS antigen of P multocida type A) The
ELISA test conditions were kept same as
standardized for P multocida B:2 antigens
For a type of antigen, all optimum dilutions of The coating antibody, HS-1 Mab and The conjugate were kept same as standardized for
that type of P multocida B:2 antigen The
whole killed bacterial cell antigen was used as undiluted stock of the bacterial culture while sonicated and the LPS antigen was used in dilution 1:5 Though, amount of protein in sonicated antigen, and carbohydrate in the
LPS antigen of P multocida type A culture
broth were not estimated but the number of cfu were estimated and the dilution of the stock culture in BHI broth was so adjusted that the number of cfu in the BHI broth of
both P multocida B:2 and P multocida type
A became similar in number This was done
to have amount of protein in sonicated antigen and carbohydrate in LPS almost similar to the
antigen of P multocida B:2 The number of cfu in stock culture of P multocida type A were higher than that in stock culture of P
multocida B:2 The stock culture of P multocida type A was, therefore, appropriately diluted and then the whole killed bacterial cell, sonicated and the LPS
Trang 8antigens of P multocida type A were
prepared respectively, for P multocida B:2
Four replicates of a type of the antigen of P
multocida type A were tested together
positive control In positive control, another
anti-pasteurella multocida monoclonal
antibody that cross reacts equally with P
multocida B:2 and P multocida type A
(Pankaj Kumar,2014) was used The dilution
of the cross reacting monoclonal antibody
was kept similar to the dilution of HS-1 Mab
standardized for a type of antigen
Normalization of data
To minimize test to test variations,
normalization of the data was done The OD
value of test well could either be converted as
percentage of reaction in comparison to OD
value of known positive control or it could be
expressed as ratio of test well OD value over
negative control well OD value In the study,
ratio of test well OD over mean +3SD value
of negative control wells OD was used for
normalization of data
Evaluation of the test for clinical diagnosis
of HS
The ELISA test standardized and optimized
with laboratory prepared P multocida B:2
antigens was assessed for its fitness as test for
clinical diagnosis of the disease The test was
evaluated not only for its fitness for clinical
diagnosis of HS but also its capacity to
diagnose the disease at an early stage, before
the appearance of clinical symptoms The
bacteria could be in any of the three forms i.e
whole bacterial cell, disintegrated bacterial
cell and LPS The sonication mimicked the
disintegration of the bacterial cell While,
whole bacterial cell is expected in the early
stages of the disease, protein and LPS
antigens of disintegrated bacteria are likely to
be present in samples collected from animals
with clinical symptoms or died of HS The
ELISA test was developed for all three types
of antigens but the ELISA test developed for whole killed bacterial cell was used This was done to assess the capacity of The ELISA test for early diagnosis of HS
A total of 9 mice were experimentally
infected with P multocida B:2 P52 strain by
s/c inoculation of 0.5 ml BHI broth containing
100 cfu One mouse was kept as non-infected control The control mouse received 0.5ml
BHI broth without P multocida B:2
The heart blood of the infected mice was collected at 2, 4, 8, 12 and 24 h post infections The surviving mice were killed by cervical dislocation and heart blood was collected directly by puncturing the heart Two mice were used at each sampling except The heart blood of the non-infected mouse was collected at the time of sampling at 24 h post infection
Isolation of bacteria from clinical samples
The Isolation of bacteria, ELISA test and PCR were performed on the collected blood samples The control mouse was killed by cervical dislocation The blood samples at various time post infections were tested by The ELISA test as standardized for whole killed bacterial cell was setup The ELISA test results were compared with “Gold standard’ of HS diagnosis The heart blood samples of various time post infections were tested for isolation of bacteria and for the detection of the bacterial DNA by PCR Heart blood of individual infected mouse, at various sampling, was streaked on a 35 mm 5% sheep blood agar Petri plate for isolation of the bacteria and the heart blood of two mice at a sampling time was pooled and then used for detection of the target bacterial DNA by PCR The pooled blood samples were also used in The ELISA test
Trang 9The ELISA test
The ELISA test was performed with the
optimum dilutions 1:1000, 1:500 and 1:800 for
the coating antibody, HS-1 Mab and The
conjugate, respectively Two dilutions of the
blood samples (Undiluted and 1:2 diluted) were
tested The heart blood collected at all the
sampling time, was hemolysed by adding
distilled water three times This was done to
remove any interference of red blood cells in
antigen binding Approximately 100µl of blood
could be collected from each mouse and it could
be suitably hemolysed with distilled water
resulting in to1:3 dilution of the blood This 1:3
diluted blood sample was used as undiluted
blood sample The mice at 24 h post infection
died much before the time of sampling (during
very early hours of morning) Very little amount
of heart blood could be collected As such there
was no blood sample, therefore heart was cut
and little heart blood samples whatever amount,
could be collected by washing the thoracic
cavity with 0.3 ml distilled water The prior
dilution of this heart blood sample of 24 h in
undiluted blood, could not be determined The
samples appeared to be highly diluted (Fig 1
and 2) The layout of ELISA well module is
shown in Figure 11 and OD values of heart
blood samples are shown in Table 1
The PCR
The PCR was performed on undiluted as well as
diluted blood samples (Table 2) The P
multocida gene specific PCR performed on
direct blood samples showed positive
amplification in the samples collected 4 and 8 h
post infection No amplification was recorded
for heart blood sample of 12 and 24 h post
infection
Detection of target DNA in infected mice
blood by PCR
P multocida gene specific, as well as duplex
PCR for amplification of P multocida type
and P multocida B:2 were set up P
multocida, serogroups B and type B:2 specific
multiplex-PCR was used to confirm the
strains as P multocida type B:2 Different
sets of primers specific to capsule as well as 16s RNA were used as mention in the (Table 3)
PCR parameters
Three PCR were performed i.e simplex, duplex and multiplex Simplex PCR using KMT1T7 and KMT1SP6 set of primer
specific to P multocida, duplex PCR for simultaneous detection of P multocida Type
B using two sets of primer i.e
KMT1T7/KMT1SP6 and KT SP61 /KTT72
however, multiplex PCR using three sets of
primer for simultaneous detection of P
multocida Type B:2 were used
The PCR was performed in a thermocycler (Veriti, Invitrogen) with a total reaction volume of 25 µL using KAPA blood PCR kit (KK7003)
Results and Discussion
Culture of P multocida
The bacteria were streaked on 5% sheep blood agar Petri plate Characteristic translucent dew drop like colonies appeared after incubating overnight at 35-36oC Gram’s staining of the bacterial smear showed Gram negative small cocco-bacilli appearance Presence of only gram negative small cocco-bacilli bacteria in the smear, indicated purity
of the culture (Fig 3)
P multocida was identified by various
biochemical tests (Table 3) and P multocida type B was confirmed by PCR The PCR
showed amplification of target DNA (Fig 4) showing a band of amplicon size of ~620 base pairs (bp)
Trang 10Preparation of bacterial antigens
No bacterial growth was observed, after
overnight incubation of P multocida B:2
broth
Estimation of colony forming units
The number of colonies on the Petri plate was
30, 24 and 27, inoculated with dilution 10-12
of the broth of bacterial culture (Fig 5) and
therefore the Petri plates of 10-12 gave an
average of 27 bacterial colonies
The BHI broth of the bacterial culture
contained 27X10-12 cfu in 50µl or 54X10-13
cfu per ml of The P multocida type B:2 BHI
broth
This was expected to be an accurate
estimation of the bacterial antigen because the
bacterial culture was terminated at 8 h, during
log phase of the bacterial growth In over
grown bacterial culture, the culture might
contain dead bacterial cell and cfu may not be
the correct estimation of whole cell bacterial
number
Estimation of protein concentration of
sonicated antigen
The standard curve (Fig 6) was made using
bovine serum albumin with concentrations of
0, 10, 20, 30, 40, 50 µg/ml for the microassay
(extinction coefficient of BSA is 0.667)
Protein concentration of sonicated antigen
was estimated from standard curve with
absorbance at 595nm It was estimated to be
44 µg/ml
Estimation of carbohydrate concentration
of LPS antigen
The stock concentration of LPS antigen
estimated by sulphuric acid-UV method was
1.392g/ml
Assay development and validation
Sandwich ELISA was standardized for all three types of antigens by performing Chequer board titration to determine the optimum dilutions of: 1-The coating antibody, 2-HS-1 Mab and 3-The conjugate The optimum dilutions of antigens were taken as 1:5, 1:50 and 1:100
Chequer board titration for whole killed bacterial cell antigen
Raw OD values recorded for antigen concentration 1:5, 1:50 and 1:100 in Chequer board titrations are shown in (Tables 4, 5 and 6), respectively
The ELISA plate OD values for antigen dilution 1:50 were used for determination of optimum dilutions The Optimum dilutions of bio-reagent were selected as the coating antibody- 1:1000, HS-1 Mab-1:500 and the conjugate-1:800
Chequer board titration for sonicated antigen
Raw OD values recorded in Chequer board titrations for antigen concentration 1:5, 1:50 and 1:100 are shown in (Tables 7, 8 and 9), respectively The ELISA plate OD values for antigen dilution 1:100 were used for determination of optimum dilutions The Optimum dilutions of reagent were selected as: The coating antibody- 1:1000, HS-1 Mab-1:1500 and the conjugate-1:6400
Chequer board titration for LPS antigen
Raw OD values recorded for antigen concentration 1:5, 1:50 and 1:100 are shown
in (Tables 10, 11 and 12), respectively
The ELISA plate OD values for antigen dilution 1:5 were used for determination of
Trang 11optimum dilutions The Optimum dilutions of
reagent were selected as: The coating
antibody- 1:1000, HS-1 Mab-1:1500 and the
conjugate-1:3200
Repeatability study
Three plates on three days with 24 replicates of
one antigen dilution in one plate were set up
Repeatability of the test was estimated from
calculation of coefficient of variation (CV) from
OD values The experiments were performed
for all three types of antigens and the values
of estimated CV are given in (Tables 13, 14,
15) for whole killed bacterial cell, sonicated
and LPS antigens, respectively The CV
estimated for intra-plate, inter plate and inter
days for all three antigens were largely within
20% For some ELISA plate, the values of
CV was slightly above than 20% but below
21% This was due to rounding off values
during calculation However, 23 % and as
high as 25 % was recorded for whole killed
bacterial cell antigen only
Estimation of cut off value
Cut off values were estimated for declaring
positive or negative result The mean +3SD
values calculated for all three types of antigen
and for both formats of negative control
wells, are given in (Table 16) The cut off
value for whole killed bacterial cell, sonicated
and LPS antigens were found to be 0.11,
0.086 and 0.1, respectively
Analytical sensitivity
The ELISA was performed with various two
fold dilutions of an antigen The highest
dilution of the antigen where OD value
equivalent to cut off OD values, was recorded
as ELISA titre The OD values were plotted in
MS Excel 2007 and exact dilution
corresponding to the cut off values was
estimated for the ELISA titre (Fig 7, 8 and 9)
for whole killed bacterial cell, sonicated and LPS antigen, respectively
OD values with whole killed bacterial cell, sonicated and LPS antigens are given in (Tables 17, 18 and 19) respectively The ELISA titres for whole killed bacterial cell, sonicated and LPS antigen were 1:320, 1:114 and 1:80, respectively Amount of antigen were: 5.4X1014cfu per ml, 44µg per ml and 1.392g per ml for whole killed bacterial cell, sonicated and LPS antigen, respectively Therefore, the analytical sensitivity for whole killed bacterial cell was 1.6X1011cfu/ml, 385ng/ml for sonicated antigen and 17.4mg/ml for LPS antigen
Analytical specificity
High OD values in positive control wells and
OD values close to zero in wells of P
multocida Type A bovine strain (Table 20)
established the analytical specificity of the
test for P multocida type B against most
closely related bacteria
Standardized ELISA for clinical diagnosis
1 Mab and The conjugate, respectively
Isolation of bacteria from clinical samples for evaluation of the test
The bacterial growth was observed with 20 colonies in the blood samples taken after 8 h and 45 colonies 12 h while no discrete colony was seen in the culture of blood sample of 24
h The bacteria were identified as P
multocida by appearance in Gram’s stain
smears
Trang 12The ELISA test
The ELISA test was performed with the
optimum dilutions 1:1000, 1:500 and 1:800
for the coating antibody, HS-1 Mab and The
conjugate, respectively Two dilutions of the
blood samples (Undiluted and 1:2 diluted)
were tested.The layout of ELISA well module
is shown in Figure 11 and OD values of heart
blood samples are shown in Table 21
The PCR
The PCR was performed on undiluted as well
as diluted blood samples The P multocida
gene specific PCR performed on direct blood
samples showed positive amplification in the
samples collected 4 and 8 h post infection No
amplification was recorded for heart blood
sample of 12 and 24 h post infection The PCR
for duplex amplification of P multocida B and
serotype B:2 specific gene performed on blood
samples showed positive amplification in
samples collected 4, 8 and 12 h post infection
but the lane no.7 of (Fig 10) for samples
collected 24 h post infection showed smearing
This indicated high amount of template
bacterial DNA in the sample of 24 h post
infection
The blood sample of 24 h post infection was
further three fold diluted to lower the amount
of template DNA and then PCR for duplex
amplification of P multocida B and serotype
B:2 specific gene was performed Positive
amplification could be noted in samples
collected 8, 12 and 24 h post infection The
sample of 8 and 12 h post infection showed
amplification for both P multocida B and
sero type B:2 specific genes The samples at
24 h post infection showed amplification of
only P multocida serotype B:2 specific gene,
lane no.6 of (Fig 10) The target bacterial
DNA, therefore, could be detected in heart
blood samples of 4, 8, 12, and 24 h post
infection
isolation, ELISA and PCR for the samples
of experimentally infected mice
The results of isolation of bacteria, The ELISA test and PCR on heart blood samples
of experimentally infected mice are given in (Table 20 and 21)
The ELISA recorded positive results even in 2
h post infection but PCR tested positive in 4 h sample and bacteria started appearing at 8 h post infection The OD values in test wells of infected blood samples demonstrated much higher value when compared with heart blood
of non-infected mouse The OD values in both undiluted and diluted heart blood samples demonstrated an increasing trend from 2 h onwards to 12 h However, lower
OD values were recorded in samples of 24 h post infection
Prompt diagnosis of bacterial infectious diseases of livestock is an important step in management of outbreaks In many developing or underdeveloped Asian countries, including India, an effective vaccination based control programme is not in place This is due to poor animal husbandry conditions as a result of lack of facilities, non-cooperative attitude of livestock owners due
to ignorance and poor knowledge For diseases that occur on an outbreak scale and
an effective vaccine is also available for their successful prevention, even then veterinary health personnel are compelled to adopt approach of treatment of diseased animals with antibiotics In India, this approach of antibiotics treatment and not prevention of the disease by regular vaccination with wider coverage is a common practice for tackling
HS Per acute nature of the disease results in
to very short course of the disease and the fact that antibiotic treatment is disadvantageous at later stage of the disease, there is very little time available with veterinarians to start the
Trang 13treatment of diseased animals with antibiotics
Treatment with antibiotics is of limited value
unless carried out in the very early stages
(Buxton and Fraser, 1977) Huge economic
losses, predominantly, due to death of young
adult buffaloes creates a panic and often
antibiotic treatment is started without
confirmation of the disease The
indiscriminate use of antibiotics leads to
emergence of resistance in bacteria, undue
contamination of the environment and the life
of the animal is also not saved and money of
livestock owner is wasted on purchase of
expensive antibiotics It is therefore demand
of time that, sensitive, specific, point-of care
and affordable diagnostics should be available
to veterinarians
Importance of diagnostics led to focus of
researchers on developing newer and better
diagnostics This resulted in to rapid
advancement in techniques and technology of
disease diagnosis
In the present study, a monoclonal antibody
based sandwich ELISA was developed for
HS An anti-pasteurella multocida B:2 strain
P52 (vaccine strain) whole bacterial cell
polyclonal serum was used as capture
antibody The immune response to whole
bacterial cell involved the activation of
multiple B-cell which target a specific epitope
on the bacterial cell As a result, a large
number of antibodies were produced with
different specificities and epitope affinities In
contrast, monoclonal antibodies are antibodies
produced by a single B lymphocyte clone As
most monoclonal antibodies lose some or all
of their binding affinity when adsorbed on
plastic, there is an advantage in coating with
polyclonal antibodies because at least some of
the population of antibodies retain binding
activity when adsorbed (Wild and Kusnezow,
2005) Considering the advantages of
polyclonal serum as capture antibody, the
anti- pasteurella whole bacterial cell
polyclonal serum was used as capture antibody and the HS-1 Mab was used as tracing antibody Though, the polyclonal
serum would capture P multocida or other bacterial antigen close to the P multocida,
however, specificity of the test would be maintained The tracing monoclonal antibody,
being P multocida B specific, would bind only to captured P multocida type B
The objective of the research was not only to develop a specific ELISA for HS but the test should have the capacity for early diagnosis
of the disease For this reason, the standardized ELISA was applied on samples collected at different hours post infection of experimentally infected mice The blood samples of as early as 2 h post infection were tested for presence of the bacterial antigen The clinical manifestations of the typical HS disease caused by B:2 or E:2 strains include a rise in temperature, respiratory distress with nasal discharge, and frothing from the mouth, leading to recumbency and death Septicemia
is the characteristic feature in all the disease conditions The incubation period varies from
3 to 5 days In peracute cases, sudden death with observable clinical signs may be observed (Carter and De Alwis, 1989) and De Alwis (1992b) Characteristic of sudden onset
of disease leading to rapid death of infected animals is similar to that seen in other clinical conditions known to involve endotoxic shock
(Radostits et al., 2007) Horadagoda et al.,
(2001) studied role of endotoxin in the pathogenesis of hemorrhagic septicemia in buffaloes The findings demonstrated that a progressive endotoxaemia and associated sequel, correlates with the development of overt hemorrhagic septicemia disease and sudden death in buffaloes It was, therefore, expected that the antigens of three types i.e whole bacterial cells, disintegrated bacterial cells due to death of bacteria and the LPS released from the dead bacterial cells are likely to be present in the clinical sample In
Trang 14view of this, ELISA was standardized for
whole killed bacterial cells, sonicated
bacterial cells antigen and the LPS antigen
During the standardization of ELISA, it was
observed that The ELISA test, with sonicated
antigen performed better than whole killed
bacterial cell and the LPS antigen because, for
sonicated antigen, the ELISA plate OD values
for antigen dilution 1:100 were used for
determination of optimum dilutions The
optimum dilutions of reagent were selected
as: The coating antibody- 1:1000, HS-1 Mab-
1:1500 and the conjugate-1:6400 Though, for
the LPS antigen, higher optimum dilutions of
bio-reagents: The coating antibody-1:1000,
HS-1Mab-1:1500 and The conjugate-1:3200
were selected but, the test for LPS could be
performed with 1:5 dilutions of antigen only
and the OD value of selected well was also
0.206, just reaching to the minimum
acceptable OD value as describe in section
3.6.3.1 For sonicated antigen, the selected
antigen dilution was 1:100 and the OD value
of the selected well was 0.840, much higher
than the OD value of 0.206 for LPS antigen
The physical form of the antigen influences
how one detects its reaction with an antibody
If the antigen is a particulate, one generally
looks for agglutination of the antigen by the
antibody If the antigen is soluble one
generally looks for the precipitation of the
antigen after the production of large insoluble
antigen-antibody complexes (Mayer, 2013)
Lower performance of the test for whole
killed bacterial cell antigen was, therefore,
due to physical form of the antigen and the
soluble form of antigens in sonicated and the
LPS antigen preparations performed better
The ELISA test could perform only in lower
dilution of the LPS antigen (1:5 dilution)
This was, perhaps, due to lower amount of the
antigen in the LPS antigen preparations The
epitope of the HS-1 Mab has been reported to
be present on fimbriae (Pankaj Kumar, 2014)
Extraction of LPS from bacterial cells would
have been satisfactory, but the epitope of the HS-1 Mab is not present on LPS or outer membrane proteins co-extracted with LPS
Purified LPS of P multocida has been
reported to be non protective and it was concluded that LPS preparation contaminated with outer membrane protein could afford protection (Muniandy and Mukkur, 1993) It appeared that, some amount of fimbrial antigen was co-extracted with the LPS Being
a soluble antigen, it could perform better in ELISA and therefore, higher optimum dilutions of bio-reagents were selected but being low in amount in LPS antigen preparation, it could work only in lower dilution of the LPS antigen
The test is considered a repeatable test if CV
is within 20% The ELISA test was found to
be a repeatable test with few exceptions when
CV varied more than 20% For most ELISA test the CV recorded beyond 20% in decimals only and it was due to rounding off during calculations In some instances, as high as 23% and 25% CVs were also recorded However, higher percentages of CVs were not due to higher values of standard deviations but due to lower mean values The CV is calculated by dividing SD by mean value The test could be considered a poorly repeatable test only if value of standard deviation is high Analytical sensitivity for whole killed bacterial cell was 1.6X1011cfu/ml, 385ng/ml for sonicated antigen and 17.4mg/ml for LPS antigen The analytical sensitivity of whole killed bacterial cell and the sonicated antigen are of more relevance for standardization of test as diagnostic test of HS This is because
of the fact that the fimbrial antigens are likely
to be present in whole killed bacterial cell and
in sonicated antigen preparations
It has been reported that 1.6X109 cfu
Escherichia coli (Tanner, 1948) would weigh
a gram and therefore, 385ng of the sonicated